The present invention relates generally to a method and apparatus for determining the concentrations of lipoproteins in blood using a continuous flow analyzer.
It is well-known that total serum cholesterol is strongly correlated with the incidence of atherosclerosis and coronary heart disease. More recent studies also indicate that specific fractions of cholesterol are more closely associated with coronary heart disease than others. Recent studies have implicated LDL (low density lipoprotein) as the class of lipoprotein responsible for the accumulation of cholesterol in cells, whereas HDL (high density lipoprotein) has been shown to be important in the removal of excess cholesterol from cells. Thus, increased levels of LDL cholesterol have been associated with the greater risk of coronary heart disease, while a strong inverse relationship exists between HDL cholesterol and the risk of coronary heart disease.
In addition to LDL and HDL, several other lipoproteins have been shown to represent independent risk factors for coronary heart disease. Increased plasma concentrations of lipoprotein(a) [Lp(a)], a cholesterol rich lipoprotein, has been observed in survivors of myocardial infarction. One study, which reports the relationship of levels of Lp(a) and coronary heart disease in patients who underwent coronary angiography, concluded that plasma Lp(a) appears to be a major independent coronary risk factor with an importance approaching that of LDL cholesterol or HDL cholesterol.
Increased levels of intermediate density lipoprotein (IDL) and cholesterol rich very low density lipoproteins (VLDL) are also associated with the progression of coronary heart disease. Elevation of plasma VLDL is seen in survivors of myocardial infarction, suggesting the possible involvement of this lipoprotein in the atheroschlerotic process.
Measurement of total cholesterol alone may not be adequate to identify subjects at risk for coronary heart disease. An individual with normal or near normal levels of total cholesterol may still be at risk because of low HDL levels, elevated Lp(a) levels, or elevated levels of VLDL. Moreover, the predictive power of total cholesterol for risk of coronary heart disease diminishes in men with increasing age. Therefore, assessment of the distribution of cholesterol among all the lipoproteins (a lipoprotein cholesterol profile), in addition to total cholesterol, is desirable in order to accurately assess risk for coronary heart disease.
Methods currently used to determine the concentration of cholesterol in the different lipoprotein classes can be divided into direct methods and indirect methods. In direct methods, lipoprotein cholesterol is determined by enzymatic assay of the individual lipoproteins, which are separated by ultracentrifugation, electrophoresis, or selective precipitation. The most accurate of these methods involves ultracentrifugation. However, ultracentrifugation separation methods are expensive, time-consuming, and are not practicable for clinical applications wherein multiple analyses are carried out in large numbers.
Indirect methods, as a general rule, are better suited for clinical applications than are direct methods. The most commonly used method for measurements of lipoprotein cholesterol performs multiple analyses using different aliquots of the same plasma sample. Total cholesterol (TC) is measured using a first aliquot of the sample. In a second aliquot, VLDL and LDL are removed by precipitation and the supernatant is assayed for cholesterol to provide a measure of HDL cholesterol. An estimate of VLDL is obtained by measuring the triglycerides (TG) in a third aliquot using the Friedewald formula or is measured directly after ultracentrifugal isolation of very low density lipoprotein. The LDL cholesterol concentration is not measured directly, but is calculated by subtracting the HDL cholesterol and VLDL cholesterol values from the total cholesterol.
Although this method is relatively rapid and inexpensive, there are several steps where error could be introduced. For example, accurate measurements of HDL depends on complete precipitation of apo-B containing lipoproteins. Traces of LDL in the supernatant can lead to overestimation of HDL cholesterol. Moreover, the multiple assumptions involved in the Friedewald formula make this method susceptible to error. In addition, this method does not provide a separate measure of IDL cholesterol or Lp(a) cholesterol. Instead, these values are included in the LDL cholesterol measurement.
The VAP method (Vertical Auto Profile) provides a direct method for determination of lipoprotein concentrations. The VAP method uses short spin density gradient vertical ultracentrifugation to separate the classes of lipoproteins. Analysis of cholesterol is made using an air segmented continuous flow analysis system to provide a lipoprotein cholesterol profile. The VAP method provides a direct measure of lipoprotein cholesterol using a single aliquot of plasma. However, VAP requires a relatively large sample (1.3 ml), and the equipment used in the VAP method is cumbersome, making its operation and maintenance difficult. Furthermore, this method causes overlapping of adjacent lipoprotein peaks in the cholesterol profile resulting in a substantial loss of resolution. Quantification of Lp(a) and IDL, which are not well-separated from other lipoproteins by density-gradient centrifugation becomes difficult particularly when present in small amounts.
Another method for determination of cholesterol distribution among plasma lipoproteins involves the separation of lipoproteins by high performance liquid chromotography and the on-line detection of cholesterol in the postcolumn effluent using an enzymatic reagent. This method also provides a direct measure of lipoprotein cholesterol. However, this method requires a relatively long retention period for separation of the sample. Moreover, the separation technique results in some loss of lipoproteins which could result in an underestimation of cholesterol concentration.
Accordingly, there is a need for a high rate method for direct determination of cholesterol distribution among plasma lipoproteins which is suitable for clinical applications.